CRISPR-Cas in Acinetobacter baumannii Contributes to Antibiotic Susceptibility by Targeting Endogenous AbaI

ABSTRACT Acinetobacter baumannii is a well-known human opportunistic pathogen in nosocomial infections, and the emergence of multidrug-resistant Acinetobacter baumannii has become a complex problem for clinical anti-infective treatments. The ways this organism obtains multidrug resistance phenotype include horizontal gene transfer and other mechanisms, such as altered targets, decreased permeability, increased enzyme production, overexpression of efflux pumps, metabolic changes, and biofilm formation. A CRISPR-Cas system generally consists of a CRISPR array and one or more operons of cas genes, which can restrict horizontal gene transfer in bacteria. Nevertheless, it is unclear how CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii. Thus, we sought to assess how CRISPR-Cas affects biofilm formation, membrane permeability, efflux pump, reactive oxygen species, and quorum sensing to clarify further the mechanism of CRISPR-Cas regulation of Acinetobacter baumannii antibiotic resistance. In the clinical isolate AB43, which has a complete I-Fb CRISPR-Cas system, we discovered that the Cas3 nuclease of this type I-F CRISPR-Cas system regulates Acinetobacter baumannii quorum sensing and has a unique function in changing drug resistance. As a result of quorum sensing, synthase abaI is reduced, allowing efflux pumps to decrease, biofilm formation to become weaker, reactive oxygen species to generate, and drug resistance to decrease in response to CRISPR-Cas activity. These observations suggest that the CRISPR-Cas system targeting endogenous abaI may boost bacterial antibiotic sensitivity. IMPORTANCE CRISPR-Cas systems are vital for genome editing, bacterial virulence, and antibiotic resistance. How CRISPR-Cas systems regulate antibiotic resistance in Acinetobacter baumannii is almost wholly unknown. In this study, we reveal that the quorum sensing regulator abaI mRNA was a primary target of the I-Fb CRISPR-Cas system and the cleavage activity of Cas3 was the most critical factor in regulating abaI mRNA degradation. These results advance our understanding of how CRISPR-Cas systems inhibit drug resistance. However, the mechanism of endogenous targeting of abaI by CRISPR-Cas needs to be further explored.

.The importance section is a little bit redundant, it can be cut shorter or deleted since the content is somehow covered in abstract and discussion. .Line 63-89, the introduction of CRISPR-Cas is not in a very good order and may lead to confusion. Try starting with overall introduction of CRISP-Cas in bacteria immune system, then lead to I-Fb CRISPR-CAS and I-Fb CRISPR-CAS3 .Line 91, the majority of strains do not have the presence of CRISP-Cas, so this title might be mislead. .Line 95, define MDR (resistant to three or more classes of antibiotic)and sensitive. .line 112, if you want to show no differences between group, an endpoint statistical test is needed. .Line 114, the supplement table S3 which should support the conclusion is not well-organized and with no table title or  description. You may reorganize the table S3 or only make conclusion draw from table 1. .line 139 Can't draw conclusion about drug resistance here, only resistance gene expression level .line 160 figure 4B and 4C clarify which genes are related to efflux pump and which are related to TCS .line 235 From figure 7F and 7G, if the ANOVA test of the two figure are done separately, the significancy can not be combined to get 62.5%. .line 240-244, the explanation for figure 8 is too vague, hard to understand what those gene in 8E-8L are. This paragraph is too general. .line 258 There is no figure 9. .line396 Is there a washing step for permeability assessment? .Line 765 I represents? .Line 838 Why separate 7F and 7G? Was the ANOVA test done separately in 7F and 7G?
Reviewer #2 (Public repository details (Required)): A whole genome sequence of A. buamannii strain AB43 has been submitted to GenBank with the accession number CP083181.
A transcriptome sequencing data has been submitted to NCBI with the accession number SRR17299399.
Reviewer #2 (Comments for the Author): In this manuscript, authors verified the function of CRISPR-Cas system in rendering A. baumannii antibiotic sensitivity through various ways, such as inhibiting hydrolyzing enzymes, efflux pumps and biofilm formation, production of ROS and H2O2, decreasing the activity of SOD and NADP. However, the reason of the above changes is concluded to the regulation axis of Cas3 -AbaI -efflux pumps. It seems not very persuasive that there is not enough evidence to prove that Cas3 is the core of CRISPR-Cas system to lead to the outcomes, since the deletion of other components in this system could also affect the expression of Cas3 and bring similar results. Moreover, changes of AbaI and efflux pumps are not the only reason for antibiotic sensitivity of A. baumannii. So, the function of CRISPR-Cas is not clearly explained by the current results.
General comments: The manuscript is difficult to follow, many of the sentences need revision, and English needs an extensive proofreading.
The results section includes a discussion of results, while the discussion section reports a repetition of results. In addition, the discussion section does not fully explain all the results obtained in this work. Both sections need to be revised.
There is a common problem in the manuscript including figures and tables, that gene names should be all lowercase in italic, and protein names should be capitalized for the first letter and non-italic. Take AbaI as an example, its gene name is abaI, and protein name is AbaI. Misused of the names are all through the manuscript.
Specific comments: In some sentences, the expression is not rigorous. For example, in line 17-20, the factors of altered targets, decreased permeability, overexpression of efflux pumps, metabolic changes and biofilm formation could cause resistance phenotype in A. baumannii, but not the way of obtaining multidrug resistance genes. It's better to say "obtains multidrug resistance phenotype".
In line 26 and 65, authors say AB43 has a complete I-Fb CRISPR-Cas system. Please clarify what is the type of I-Fb, since according to the references, the type I CRISPR-Cas system are classified as Type I-F1, Type I-F2 (Ref. 25). Please use the official classification name and provide correct references.
In line 79-80, the authors describe that QS activates type I-F CRISPR-Cas expression and CRISPR adaptation in P. aeruginosa. And in line 84-85, authors say that the purpose of this work is to investigate the role of the Type I-Fb CRISPR-Cas system in modulating QS operation in A. baumannii. What's the relationship between QS and Type I-Fb CRISPR-Cas system? Who regulates who? Or are they in a regulation circle? There should be some background about this knowledge.
Between line 91-101, please list the analysis of antibiotic susceptibility results of all the 245 clinical strains. The number/percentage of MDR and sensitive strains in 132 CRISPR-Cas negative isolates should also be shown in the results.
Line 98-101, should it be related between MDR and missing of component of the CRISPR-Cas system? Since most of the strains with incomplete or without CRISPR-Cas system are MDR.
In Figure 1B, the subscripts of X axis are wrong, they should be I-Fa and I-Fb. Please also check whether they should be I-F1 and I-F2.
In line 117-118, authors say that WT and all the gene rescue mutants were susceptible to all the 24 antibiotics. However, in Table 1, AB43 and the complement strain are resistant to piperacillin, and intermediate to cefotaxime and ceftriaxone. Please  correct the results. Line 124, blaOXA-51-like should be blaOXA-51-like. Moreover, as shown in the results that the expression of ampC and blaOXA-51-like were increased significantly in the ΔCRISPR-Cas mutant, why don't authors consider the inhibition function of CRISPR-Cas on these two enzymes, but give the conclusion only on regulation of efflux pumps?
Line 193-195, the explanation sounds conflict with the results. Since dysfunction of the CRISPR-Cas system could enhance biofilm formation, and at the same time dampen bacterial permeability, both two results will result in the increased resistance to antibiotics, thus show a synergistic effect with efflux pumps, but not limit the efflux of antibiotics.
Line 240, authors say that several drug resistance factors are under that control of AbaI according to ref. 47. However, AbaI is not mentioned in this reference. BTW, authors did not say which factors are under the control of AbaI, thus the expression results could hardly be explained.
In Figure 8F, the abaI expression is increased in AB43ΔCRISPR-Cas mutant, that means CRISPR-Cas system represses the function of AbaI. And in Figure 8I, adeB expression is increased in AbaI knockout mutant, which shows another negative regulation. In this case, downregulation of CRISPR-Cas could result in an upregulation of abaI, and finally lead to a decrease of adeB expression. However, in Figure 4C, the expression of adeB is increased for about 5 folds in ΔCRISPR-Cas mutant. How to explain the conflict results? Moreover, if the results in Figure 8 are correct, expression of CRISPR-Cas will inhibit the activity of AbaI, which will boost the expression of AdeB, but not lead to ABC transporter inhibition as mentioned by authors in line 258. In addition, in line 257-258, authors describe the relationship among Cas3, AbaI and ABC transporter, but all the qPCR results are obtained in ΔCRISPR-Cas mutant, and there is no evidence about the changes of abaI and adeB expression in Cas3 knockout mutant.
There are many mistakes in the manuscript, for example, ATCC 19606 is written as ATTC 19606 in line 346; nine types of antimicrobial agents are used in the work, but is written as nine antimicrobial agents in line 363.
Line 367-377, the knockout protocol is not written in the actual way of procedure.
At the first appearance of an abbreviation, the full name should be provided, such as OD. Besides, the description of OD600 is not equal in the manuscript that it is also written as OD600nm and OD600 in some sentences.
Line 402, for the unit of NPN, should it be 10 μm or 10 μM? μl and μL are mixed used.
When writing the P value, P should be in italic. Moreover, sometimes it is shown in uppercase, but sometimes in lowercase.
Line 487, "μg ml−1" is a wrong way of showing the unit.

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In this manuscript, authors verified the function of CRISPR-Cas system in rendering A. baumannii antibiotic sensitivity through various ways, such as inhibiting hydrolyzing enzymes, efflux pumps and biofilm formation, production of ROS and H2O2, decreasing the activity of SOD and NADP. However, the reason of the above changes is concluded to the regulation axis of Cas3 -AbaI -efflux pumps. It seems not very persuasive that there is not enough evidence to prove that Cas3 is the core of CRISPR-Cas system to lead to the outcomes, since the deletion of other components in this system could also affect the expression of Cas3 and bring similar results. Moreover, changes of AbaI and efflux pumps are not the only reason for antibiotic sensitivity of A. baumannii. So, the function of CRISPR-Cas is not clearly explained by the current results.

General comments
The manuscript is difficult to follow, many of the sentences need revision, and English needs an extensive proofreading.
The results section includes a discussion of results, while the discussion section reports a repetition of results. In addition, the discussion section does not fully explain all the results obtained in this work. Both sections need to be revised.
There is a common problem in the manuscript including figures and tables, that gene names should be all lowercase in italic, and protein names should be capitalized for the first letter and non-italic. Take AbaI as an example, its gene name is abaI, and protein name is AbaI. Misused of the names are all through the manuscript.

Specific comments
In some sentences, the expression is not rigorous. For example, in line 17-20, the factors of altered targets, decreased permeability, overexpression of efflux pumps, metabolic changes and biofilm formation could cause resistance phenotype in A. baumannii, but not the way of obtaining multidrug resistance genes. It's better to say "obtains multidrug resistance phenotype". In Figure 1B, the subscripts of X axis are wrong, they should be I-Fa and I-Fb. Please also check whether they should be I-F1 and I-F2.
In line 117-118, authors say that WT and all the gene rescue mutants were susceptible to all the 24 antibiotics. However, in Table 1 Line 124, blaOXA-51-like should be blaOXA-51-like. Moreover, as shown in the results that the expression of ampC and blaOXA-51-like were increased significantly in the ΔCRISPR-Cas mutant, why don't authors consider the inhibition function of CRISPR-Cas on these two enzymes, but give the conclusion only on regulation of efflux pumps?
Line 193-195, the explanation sounds conflict with the results. Since dysfunction of the CRISPR-Cas system could enhance biofilm formation, and at the same time dampen bacterial permeability, both two results will result in the increased resistance to antibiotics, thus show a synergistic effect with efflux pumps, but not limit the efflux of antibiotics.
Line 240, authors say that several drug resistance factors are under that control of AbaI according to ref. 47. However, AbaI is not mentioned in this reference. BTW, authors did not say which factors are under the control of AbaI, thus the expression results could hardly be explained.
In Figure 8F, the abaI expression is increased in AB43ΔCRISPR-Cas mutant, that means CRISPR-Cas system represses the function of AbaI. And in Figure 8I, adeB expression is increased in AbaI knockout mutant, which shows another negative regulation. In this case, downregulation of CRISPR-Cas could result in an upregulation of abaI, and finally lead to a decrease of adeB expression. However, in Figure 4C, the expression of adeB is increased for about 5 folds in ΔCRISPR-Cas mutant. How to explain the conflict results?
Moreover, if the results in Figure 8 are correct, expression of CRISPR-Cas will inhibit the activity of AbaI, which will boost the expression of AdeB, but not lead to ABC There are many mistakes in the manuscript, for example, ATCC 19606 is written as ATTC 19606 in line 346; nine types of antimicrobial agents are used in the work, but is written as nine antimicrobial agents in line 363.
Line 367-377, the knockout protocol is not written in the actual way of procedure.
At the first appearance of an abbreviation, the full name should be provided, such as OD.
Besides, the description of OD600 is not equal in the manuscript that it is also written as OD600nm and OD600 in some sentences.
Line 402, for the unit of NPN, should it be 10 μm or 10 μM? μl and μL are mixed used.
When writing the P value, P should be in italic. Moreover, sometimes it is shown in uppercase, but sometimes in lowercase.
Line 487, "μg ml−1" is a wrong way of showing the unit.

Comments of Reviewer 1
General comments: This is a well-written paper. The hypothesis building is very solid and make sense. The data is very well-rounded and favorably tested the hypothesis.
The concept and findings are of interest, but many opportunities exist to improve the clarity of the presentation. Especially compared to the considerable amount of data, the figure legend and the result part contain not enough information to support readers to go through those data and follow the hypothesis building.
Overall response to Reviewer 1: Thank you very much for your kind comments on our manuscript. We have carefully reviewed and re-annotated the figures and rewritten/expanded figure legends, results, and discussion. In the revised version, the new text is highlighted in yellow, and the underline indicates the revised text is to identify better the changes made to the previous version. Our point-to-point response is provided below in red text. We hope these changes improve the clarity and accuracy of the presentation.
In what follows, we would like to answer the questions you mentioned and give a detailed account of the changes made to the original manuscript.

Response:
We fully agree with the reviewer. We now redraw the figure in the revised manuscript and provide a separate one-way ANOVA test for deletion strains.

Comment 12: line396 Is there a washing step for permeability assessment?
Response: Yes. "After washing three times with PBS" (Page 23, line 444-445).

Comment 13: Line 765 I represents?
Response: Thank you for pointing this out. " I represents intermediate" ( Response: We are very sorry for our negligence in the original version. We now redraw the figure in the revised manuscript and provide a separate one-way ANOVA test for deletion strains. "Compared with AB43, more than half (5/8, 62.5%) of the deletion strains: AB43Δcrispr-cas, AB43Δcas3, AB43Δcsy1, AB43Δcsy3, AB43Δcsy4, showed significantly elevated of abaI mRNA ( Figure 6C)" (Page 12, line 220-222). Cas target abaI can also repress lipid A and biofilm formation. Finally, by targeting abaI, CRISPR-Cas also controls the most multiple drug resistance genes. (Figure 9).

General comments: In this manuscript, authors verified the function of CRISPR-
We feel sorry for the inconvenience brought to the reviewer. We have carefully reviewed, proofread, and re-annotated the figures and re-written/expanded figure legends, results, and discussion. In the revised version, the new text is highlighted in yellow, and the underline indicates the revised text is to identify better the changes made to the previous version. Our point-to-point response is provided below in red text. We hope these changes improve the clarity and accuracy of the presentation.
In what follows, we would like to answer the questions you mentioned and give a detailed account of the changes made to the original manuscript. Response: We fully agree with the reviewer. "To explore whether drug resistance in A.
baumannii possessing an incomplete CRISPR-Cas system is associated with a specific Cas protein, we statistically analyzed the relationship between drug resistance phenotypes and cas genes in these 113 CRISPR-Cas-positive strains, and the results are shown in Table 1. We found that all cas genes-negative strains had significantly higher resistance rates than positive strains. I-Fa csy3-negative or I-Fb cas3-negative had the highest resistance rates in I-Fa and I-Fb cas genes-negative strains, respectively. In this regard, it is speculated that the incomplete CRISPR-Cas system, especially the loss of I-Fa csy3 and I-Fb cas3, may affect antibiotic resistance in A. baumannii." (Page 7, line 116-124) Figure 1B, the subscripts of X axis are wrong, they should be I-Fa and I-Fb. Please also check whether they should be I-F1 and I-F2.

Comment 6: In
Response: Thank you so much for your careful check. We have made corrections.
According to the reference, the type I-F CRISPR-Cas system is classified as Type I-F1, Type I-F2, also known as Type I-Fa, Type I-Fb (13). (Figure 1D Furthermore, in the Discussion section, we added the discussion about the inhibition function of CRISPR-Cas on ampC and blaOXA-51-like. "CRISPR-Cas also represses drug-resistant related genes by targeting abaI. β-lactamase is an effective resistance mechanism of A. baumannii that can inactivate β-lactam antibiotics (14). Based on the sequence homology, β-lactamases were classified into four types: class A extendedspectrum β-lactamases (ESBL), class B Metallo-β-lactamases (MBL), class C βlactamases (AmpC) and Class D β-lactamases (OXA) (15). All four types of βlactamases were reported in A. baumannii (16). Compared to AB43, various resistance genes were expressed at elevated levels in AB43Δcrispr-cas. It is striking that the intrinsic drug resistance gene ampC, often found in A. baumannii from China (17,18), can be up to 200-fold. Another intrinsic drug resistance gene, blaOXA-51-like was found up to 80-fold. Nevertheless, the expression of ampC did not change and blaOXA-51-like significant reduction in AB43ΔabaI and AB43Δcrispr-cas-abaI. A similar phenomenon has been previously reported. According to Dou et al., AHLs generated by A. baumannii might increase the expression of drug-resistance genes such as blaOXA-51-like, ampC, adeA, and adeB (19)." (Page 19, line 351-363) Comment 9: Line 193-195, the explanation sounds conflict with the results. Since dysfunction of the CRISPR-Cas system could enhance biofilm formation, and at the same time dampen bacterial permeability, both two results will result in the increased resistance to antibiotics, thus show a synergistic effect with efflux pumps, but not limit the efflux of antibiotics.
Response: Thank you for pointing this out. We have changed "Taken together, these results indicated that dysfunction of the CRISPR-Cas system could enhance AB43 biofilm biomass and dampen bacterial permeability, which in turn might limit the efflux of antibiotics." to "These results indicated that dysfunction of the CRISPR-Cas system could enhance AB43 biofilm biomass and dampen bacterial membrane permeability, which shows a synergistic effect with efflux pumps." (Page 11, line191-

Response:
We are sorry for the inappropriate citation. We have corrected and cited the above studies. "Moreover, the QS system regulates bacterial luminescence, toxin production, disinfectants tolerance, motility, biofilm formation, spore formation, and drug resistance (20)." (Page 12, line 213-215) Figure 8F, the abaI expression is increased in AB43ΔCRISPR-Cas mutant, that means CRISPR-Cas system represses the function of AbaI. And in Figure   8I, adeB expression is increased in AbaI knockout mutant, which shows another negative regulation. In this case, downregulation of CRISPR-Cas could result in an upregulation of abaI, and finally lead to a decrease of adeB expression. However, in Figure 4C, the expression of adeB is increased for about 5 folds in ΔCRISPR-Cas mutant. How to explain the conflict results?

Comment 11: In
Response: Thank you for your rigorous consideration. We also added explanations about this conflict results in the Discussion section. "The qRT-PCR results demonstrated that in AB43ΔabaI and AB43Δcrispr-cas-abaI, adeR, baeS, and some efflux pump-related genes such as adeB are increased. The results were not as expected. Additionally, the expression of adeB is increased by about five folds in AB43Δcrispr-cas mutant. The results of these two studies seem to be contradictory. This is probably that although AB43ΔabaI and AB43Δcrispr-cas-abaI were designed to be AHL-deficient, subsequent experiments with these mutants may have been influenced by the presence of abaI homologs; AbaI is similar to the LuxI family of autoinducer synthases (21). In addition, it is indicated that a combination of multiple genes may produce efflux pump phenotypes." (Page 17, line 319-327) Comment 12: Moreover, if the results in Figure 8 are correct, expression of CRISPR-Cas will inhibit the activity of AbaI, which will boost the expression of AdeB, but not lead to ABC transporter inhibition as mentioned by authors in line 258.
Response: Thank you for your significant reminding. We have changed "leading to ABC transporter inhibition because of the low level of QS ( Figure 9). " to "Furthermore, we demonstrate that the I-Fb CRISPR-Cas system may target and degrade the abaI (QS synthase) mRNA, leading to drug resistance-related biological traits and genes being inhibited because of the low level of AHLs (

Response:
We have re-written this part according to the reviewer's suggestion.
"Briefly, using AB43 genomic DNA and PKD4 as templates, the upstream and downstream homology arms of the target fragment and the kanamycin cassette fragment with FRT site were amplified, respectively. Three PCR amplicons containing overlapping regions were assembled using overlap extension PCR with specific primers (Table S4), and the resulting fragment was electroporated into competent AB43 carrying pAT04, which expresses the RecAB recombinase.
Transformants were selected on LB plates containing 7.5 μg/ml kanamycin, and PCR confirmed integration of the resistance marker. To remove the kanamycin resistance cassette, electrocompetent mutants were transformed with pAT03 plasmid, which expresses the FLP recombinase. A loss of kanamycin resistance was observed in these colonies, confirmed by PCR, and sequenced using identification primers (Table S4)